WATER-SOLUBLE FLUOROCOXIBS IN DETECTION OF CYCLOOXYGENASE-2 IN VIVO

Jashim Uddin

Biochemistry Department, Vanderbilt University, Nashville, TN 37235, USA

Abstract

Cyclooxygenase-2 (COX-2) is expressed in virtually all solid tumors and its overexpression is a hallmark of inflammation. Overexpression of COX-2 is an early event in carcinogenesis, and it plays a vital role in cancer progression, suggesting that it is a useful biomarker for both early- and late-stage cancer detection and surgical resection. Therefore, COX-2 is an ideal molecular target for visualization of inflammatory disease and a broad spectrum of human cancers. We discovered fluorocoxib A (FA), a fluorescent 5-carboxy-X-rhodamine- (5-ROX)- labeled COX-2-selective inhibitor, which has proven successful in visualization and early detection of COX-2 in inflammation and cancers in rodents and canine models including mouse intestinal polyps and skin adenocarcinomas, rat and mouse footpad inflammation, mouse tumor xenografts, and canine spontaneous bladder and oral tumors. Clinical translation of FA has a significant promise to enable early detection and accurate surgical resection of cancers in skin, colon, esophagus, and bladder. Attempts to translate FA to the clinic have been hampered by its lack of solubility in aqueous solutions appropriate for human administration. All previous administrations of FA have been in dimethyl sulfoxide or a mixed solvent consisting of DMSO, EtOH, propylene glycol, and sterile saline, that are not appropriate for human applications. We hypothesize that the biological activities of fluorocoxib A can be retained in its decarboxy analogs, and that can be used as in vivo chemical probes to selective visualization of COX-2 in cancer cells to improve early detection and delineation of surgical margin. To test this hypothesis, we synthesized N-[(rhodamin-X-yl)but-4-yl]-2-[1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl]acetamide (called fluorocoxib D), a decarboxy analog of FA. The synthesis of decarboxy-5-ROX dyes required a symmetrical condensation of 2 equiv of 8-hydroxyjulolidine with 1 equiv of 4-formaylbenzoic acid, and stirring 24h in 60% sulfuric acid at 160oC. The overall process required one Fries rearrangement and two sequential Friedel-Crafts- type electrophilic aromatic substitution reactions for the formation of the xanthene skeleton. The final conjugation required an N-succinimidyl activation of carboxylic acid group of ROX dye followed by carbodiimide coupling with INDO-butyl-anime to afford the product fluorocoxib D (FD). FD exhibited selective inhibition of COX-2 in purified enzyme assay (COX-2 IC50 = 223 nM, COX-1 IC50 > 4000 nM) and in 1483 HNSCC cells (COX-2 IC50 = 440 nM). We evaluated the feasibility of using FD for detection of COX-2 in inflammatory tissues, using established C57BL/6 mouse model of inflammation and mechanical CBA mouse model of osteoarthritis. As expected, FD enabled detection of COX-2 in footpad at 3h post-injection of the probe with high signal-to-noise ratios on Xenogen IVIS 200 imaging system. In a parallel assay, FD efficiently taken up by osteoarthritic tissues, and level of uptake was very high (20-fold) compared to contralateral non-arthritic tissues. The COX-2-selective uptake was validated by blocking the COX-2 active site using celecoxib. These results are supporting our hypothesis and allowing us to conduct further studies to establish a proof-of-principle for targeting COX-2 in pre-neoplastic and neoplastic diseases in rodents and canines.